CN102845218A - Original ecological separation method of cordyceps sinensis hirsutella sinensis - Google Patents
Original ecological separation method of cordyceps sinensis hirsutella sinensis Download PDFInfo
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Abstract
The invention discloses an original ecological separation method of cordyceps sinensis hirsutella sinensis. The original ecological separation method comprises a tissue separation method and an ascospore separation method. According to the method, seed sources are selected from fresh and ripening cordyceps sinensis in Yushu, Qinghai in China, and soil with the depth being 8 to 12 cm in a range with the diameter being 10 to 12cm around the sporocarp is remained during the collection; a culture medium is from living bodies of cordyceps sinensis host of swift moth larvae, white strong and living swift moth larvae in 4 to 5 age periods are selected, and sterile water is used for rinsing and storage and placing in time during the collection; the cordyceps sinensis and the living bodies of the cordyceps sinensis host of swift moth larvae are stored at the temperature being 0 to 4 DEG C for 0 to 15 days after the collection; the seed sources are transferred onto the culture medium for culture, and the parameters are controlled as follows during the culture: the temperature is 6 to 9 DEG C, the pH value is 6 to 6.5, the light illumination is 50 to 100lux, the air humidity is 55 to 60 percent, and the oxygen content is 20 percent.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the ecosystem partition method of the fungi-Hirsutella sinensis in a kind of Cordyceps sinensis asexual stage.
Technical background
Cordyceps sinensis is the rare treasure of integration of drinking and medicinal herbs, and centuries compares favourably with natural ginseng, pilose antler always, at home and abroad enjoys high reputation.It is not warm not dry, is particularly suitable for the old or young, sick, weak, virtual person, can take throughout the year, and the effect that has invigorating the lung and the kidney and recover one's youthful vigour, and have no side effect.But because its natural resources are deficient, price is crossed gold, and various Chinese caterpillar funguses are in succession filled and tell in market, mix the genuine with the fictitious, and are hard to tell whether it is true or false.
In order to solve disparities between supply and demand, domestic and international many scientists have carried out a large amount of research.Studies show that Cordyceps sinensis is the Clavicipitaceae Cordyceps sinensis fungus, aweto [Cordyceps Sinensis (berk.) Sacc.] is to colonize in stroma on the Hepialidae insect larvae and the compound of larva corpse.Briefly, Cordyceps sinensis is exactly by the associating of insect and fungi and give birth to.And Cordyceps sinensis fungus is found the hundreds of kind arranged, China's record also has 68 kinds, therefore, have some scholars to be commonly referred to as Chinese caterpillar fungus every by Cordyceps sinensis fungus parasitism and the fungus combination that produces fruit body.Yet, over nearly 20 years, by molecular biology and biological reducing method, proved that Cordyceps sinensis that traditional Chinese medicine is discussed only is distributed in the severe cold area of Qinghai-Tibet high height above sea level.Particularly the Cordyceps sinensis of Yushu district, Qinghai has more representativeness.Its process of growth has experienced the asexual stage to the perfect stage.And the bacterial classification in asexual stage only has Hirsutella sinensis and Cordyceps sinensis DNA fingerprint collection of illustrative plates likelihood to increase intelligence etc. up to 96 Lee %(, the molecular biology evidence of conclusive evidence Anamorph of Cordyceps Sinensis, fungus system, 19(1) 60-64,2000).Now, academia generally believe Hirsutella sinensis be the unique phorozoon of aweto (Liu's tin Jin etc., the separation in Cordyceps sinensis asexual stage and firm, the fungi journal, 1988,8(1), 35-40).And " Cordyceps sinensis " that " Fermented Cordyceps " that occur in the market especially so-called submerged fermentation is produced nearly all is not the aweto Hirsutella sinensis.Because Cordyceps sinensis bombys batryticatus body and stroma thereof are nutritious, often there are multiple other mushrooms parasitic or saprophytic, if parting material selects improper or separating method is incorrect, often may incubation growth go out different types of fungi.Especially resemble so poky bacterial classification on synthetic medium of hirsutella sinensis fungal, probably by the growth fast other fungi covered.So it is very difficult will obtaining pure aweto strain and adopt submerged fermentation method to produce Cordyceps sinensis.
Although, since first application Hirsutella sinensis partition method patent (patent No. CN85101971A) of Mr. Shen Nanying eighties, have much and occur with relevant patent with the report that the liquid deep layer fermenting of aweto mycelium is cultivated about Artificial Cultured Cordyceps Sinensis, but, up to now, the separating method of all disclosed Hirsutella sinensis is all too complicated, difficulty is large, especially synthetic medium, compare with natural ecosystem host of Cordyceps sinensis bat moth insect, there is more defective, adds culture parameters and blur, the microbiological contamination rate is high, in fact is difficult to promote.
In general, strain separating has three kinds of methods: isolation of ascospores method, tissue isolation, substrate mycelium partition method.Comparatively speaking, take front two kinds of separating methods, become more readily available pure Hirsutella sinensis.The isolation of ascospores method of Hirsutella sinensis is to utilize the ascospore of aweto maturation to sprout mycelia, obtains the purebred a kind of method of Hirsutella sinensis.The isolation of ascospores method more complicated that industry adopts (Zhang Yongjie etc. separate the Study on Diversity that can cultivate fungi from Cordyceps sinensis, and 2010, the fungus journal), thereby adopt tissue isolation more.The tissue isolation of Hirsutella sinensis is the tender tissue that utilizes the fruit body of fresh Cordyceps sinensis, under suitable medium and growth conditions, impel it to return to vegetative stage, grow up to the not mycelium of tissue differentiation, thereby obtain the purebred a kind of method of Hirsutella sinensis.So-called purebred in fact relevant with the collection ground of Cordyceps sinensis, what gather except a few peoples is the Cordyceps sinensis of Yushu district, Qinghai, and most all is Sichuan.From molecular biology, they are more or less the same, and similitude should reach more than 96%.But on quality, they have difference really.So provenance is the key factor that determines Hirsutella sinensis bacterial classification quality.Certainly, adopt what medium, it also is very important could cultivating under what conditions and obtain purebred Hirsutella sinensis.Up to the present, disclosed document comprises patent, and medium all is artificial synthetic, and employed Carbon and nitrogen sources is not the same, and ratio is also different, and compound method is with respect to natural medium more complicated all.Aspect condition of culture, described parameter area is large, for example, the control of cultivation temperature has plenty of 5～20 ℃ of (Shen Nanying, patent publication No. CN85101971A), have plenty of about 10 ℃ (Yu Yong letter patent publication No. CN1031393A) and have plenty of 5.6～9.2 ℃ (Yang Yuexiong etc., Cordyceps Militaris infects the researchs of Ghost Moths ' Larvae, 1989, zoological research).On the key link of control varied bacteria growing, described also fuzzyyer, for example, on the sterilizing part, to polypide surface sterilization 5 minutes, the experiment of Zhu Jiashi then was sterilization 7～10 minutes (Hirsutella sinensis and Paecilomyces hepiali chen DNA coexistence and competition proliferative ability, chemical composition change in the Cordyceps sinensis maturation, 2007 years to the Shen Nan diamond stone with 0.1% mercury chloride, fungus research), Yin Dinghua then adopts 75% alcohol to polypide surface sterilization (patent publication No. CN102283022A).Yet 75% alcohol surface sterilization is adopted in a large amount of experiment showed,, namely grows a large amount of miscellaneous bacterias in 2～3 days, can't eliminate at all and remove, and the growth of hirsutella sinensis fungal is covered; Even adopt concentration 0.1% mercury chloride that they were carried out surface sterilization 10 minutes, a small amount of varied bacteria growing is still arranged, work brings difficulty to later separation.Obtain Hirsutella sinensis just not saying so whether can really separate, the quality of its strain quality is also well imagined.
Summary of the invention
The objective of the invention is to overcome the shortcoming of prior art, Cordyceps sinensis pure culture---the ecosystem partition method of Hirsutella sinensis that a kind of simple, cultivation cycle is short, can obtain Yushu district, Qinghai is provided.For this reason, the present invention has taked following concrete technical scheme:
The present invention includes following steps and condition: a, provenance and be the fresh ripe Cordyceps sinensis that comes from the Qinghai Yushu Regions, will keep the soil with diameter 10～12cm, dark 8～12cm around the fruit body during collection; Medium derives from the live body of the host bat moth larvae of Cordyceps sinensis, select white and moist, healthy and strong, bat moth larvae 4～5 length of times, that live, during collection in time with rinsed with sterile water and store; The storage temperature of live body after collection of the host bat moth larvae of described Cordyceps sinensis and Cordyceps sinensis is 0～4 ℃, and the storage time is 0～15 day; B, provenance transferred cultivate on medium, parameter is controlled at 6～9 ℃ of temperature during cultivation, pH value 6～6.5, illumination 50～100lux, air humidity 55～60%, oxygen content 20%(percent by volume).
As one embodiment of the present invention, be Hirsutella sinensis isolation of ascospores method, further comprising the steps of and condition:
A, described provenance should in time use the kraft paper bag of the bacterium of going out to entangle the cordyceps sporophore part when gathering, the ascospore that the fruit body of described provenance is being launched separates, when namely on seeing brown paper, being attached with white, wire ascospore, with the ox-hide scraps of paper at wire ascospore place, sterile scissors clip surface, be stored in the PE box of the bacterium of going out for subsequent use;
B, the whole piece worm of bat moth larvae live body is immersed in the 75 degree alcohol 3 minutes, changes clothes 3 times with sterile water again; The bat moth larvae of sterilizing is cut the enteron aisle of head, crust and black, and again making beating with the little adjust pH to 6 of bar Ma Tianshou spring natural mineral water～6.5, adds 1.8～2.2%(wt%) agar, makes test tube slant or flat board, solidifies as medium;
The aseptic inoculation ring is used in c, cultivation, and the white wire ascospore on the picking ox-hide scraps of paper is transferred on described medium test tube slant or flat board, and puts into incubator and cultivated 40～45 days.
As another embodiment of the invention, be the Hirsutella sinensis tissue isolation, further comprising the steps of and condition:
A, with the polypide part of described provenance, at first fall its surperficial hymeniderm with 9～11 ℃ of sterile water wash, then, with concentration 0.1% mercury chloride they were carried out surface sterilization 10～15 minutes, then, with sterile water wash 5～7 times, be stored in the test tube of the bacterium of going out;
B, the whole piece worm of bat moth larvae live body is immersed in the 75 degree alcohol 3 minutes, changes clothes 3 times with sterile water again; The bat moth larvae of sterilizing is cut the enteron aisle of head, crust and black, and again making beating with the little adjust pH to 6 of bar Ma Tianshou spring natural mineral water～6.5, adds 1.8～2.2%(wt%) agar, makes test tube slant or flat board, solidifies as medium;
C, cultivation: the tweezers with the bacterium of going out take out Cordyceps sinensis from test tube, cut off fruit body with the aseptic operation cutter from the polypide head, use again another aseptic operation cutter that sclerotium (polypide part) is cut into 1.5～2.0 mm thin slices, transfer on medium test tube slant or flat board; Described worm slurry culture medium flat plate is put into incubator, 6～9 ℃ of set temperatures, illumination 50～100lux, air humidity 55～60%, oxygen content 20%(percent by volume) about, cultivated 50～55 days.
Beneficial effect of the present invention:
Method of the present invention compared with prior art, different from other partition methods of having reported, provenance and medium all are ecosystems, be about to fresh Cordyceps sinensis as the material source that separates the Hirsutella sinensis bacterial classification, with the fresh polypide of host's Ghost Moths ' Larvae of Cordyceps sinensis as natural medium; And condition of culture is also basic identical with the ecosystem environment that the Cordyceps sinensis of cajaput is grown, parameter is controlled at 6～9 ℃ of temperature when namely cultivating, pH value 6～6.5, illumination 50～100lux(is equivalent to indoor natural daylight), air humidity 55～60%, oxygen content about 20%.Cultivate pure female time of planting of the Hirsutella sinensis obtain with the method and shifted to an earlier date one month with respect to other partition methods, and subsequent purification, seed selection work are simpler, the highly purified excellent species of easier acquisition.The method is simple, and sterilization method is simpler, in separating incubation, does not need often to go to reject miscellaneous bacteria, particularly isolation of ascospores method, and ascospore is processed simple, and incubation time is short.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further set forth:
Fig. 1 be sclerotium cultivate the 5th day namely early stage figure;
Fig. 2 is that sclerotium cultivates the 25th day was figure in mid-term;
Fig. 3 is that sclerotium cultivates the 45th day was later stage figure
Fig. 4 be ascospore cultivate the 5th day namely early stage figure;
Fig. 5 is that ascospore cultivates the 25th day was figure in mid-term.
It is later stage figure that Fig. 6 figure ascospore is cultivated the 45th day;
Embodiment
The concrete Step By Condition of separating method of the present invention is as follows:
1, gathers the host bat moth larvae live body of fresh Cordyceps sinensis and Cordyceps sinensis
A, acquisition time are respectively after annual the Beginning of summer and before and after the Beginning of Winter; Be respectively the alpine pasture of Yushu Regions Zhiduo County, China Qinghai Province and Yushu County, height above sea level is respectively about 4500 meters and 4000 meters, the gradient is 20 °～30 °, all is in grassy marshland with gathering;
When b, the fresh Cordyceps sinensis of collection, to excavate in the lump around the soil integral body of its diameter 10～12cm, dark 8～12cm, and the on-the-spot fresh Cordyceps sinensis that will collect will be placed in the kraft paper bag of the bacterium of going out on the fruit body of fresh Cordyceps sinensis, wraps with freshness protection package and puts in the incubator that fills ice bag;
C, when gathering the host bat moth larvae live body of Cordyceps sinensis, the larva alives that the scene will collect carries out rinsing with sterile water, and places in the test tube, puts into equally in the incubator that fills ice bag, in order to transport.
2, laboratory cultures previous work
The host bat moth larvae live body of a, the fresh Cordyceps sinensis of the collection that will collect and Cordyceps sinensis is taken back the laboratory with prestissimo, and puts into rapidly in 0-4 ℃ the refrigerator, and the time is no more than 15 days.
The alcohol of the automatic incubator of b, the necessary article, the especially superclean bench that are ready to inoculating tool and other sterile workings, test tube, dull and stereotyped culture dish, multi parameters control and sterilization usefulness, mercury chloride etc.
C, the fresh Cordyceps sinensis of maturation that the chooser entity is short, polypide is sturdy, healthy and strong to guarantee hirsutella sinensis fungal, vigor is arranged, can resist preferably the sterilization of 0.1% mercury chloride long period simultaneously; Select white and moistly, healthy and strong, the host bat moth larvae live body of the Cordyceps sinensis in 4～5 length of times to guarantee to be more suitable for cultivating hirsutella sinensis fungal, also can reduce the interference of other miscellaneous bacteria simultaneously.
D, to the fresh Cordyceps sinensis of maturation, use first sterile water flush away hymeniderm, secondly with concentration 0.1% mercury chloride they were carried out surface sterilization 10～15 minutes (as far as possible surface and near parasitism or saprophytic bacteria being killed), use again sterile water wash 5～7 times, be stored in the test tube of the bacterium of going out, as the provenance of the tissue isolation of Hirsutella sinensis; There is the fresh Cordyceps sinensis of kraft paper bag to see to cover and is attached with white, wire ascospore on the brown paper, then use the ox-hide scraps of paper of sterile scissors clip surface wire ascospore comparatively dense, be stored in the PE box of the bacterium of going out, as the provenance of the isolation of ascospores method of Hirsutella sinensis.
E, to live body bat moth larvae, at first the whole piece worm is immersed in the 75 degree alcohol 3 minutes, secondly change clothes 3 times with sterile water, in case the living contaminants on the polypide.Again, the bat moth larvae of sterilizing is cut the enteron aisle of head, crust and black, collect, pull an oar with bar Ma Tianshou spring natural mineral water again, little adjust pH to 6～6.5, adding 1.8-2.2%(percentage by weight) agar, make test tube slant or flat board, wait to solidify the natural medium that namely can be used as the separation Hirsutella sinensis.
3, laboratory cultures work
A, in super-clean bench or hundred grades of aseptic operating rooms, ascospore is launched the fruit body of Sheng phase and carry out isolation of ascospores: use the aseptic inoculation ring, the white wire ascospore on the picking ox-hide scraps of paper is transferred on worm slurry medium test tube slant or flat board.
B, in super-clean bench or hundred grades of aseptic operating rooms, the fresh Cordyceps sinensis of sterilizing is organized separation: the tweezers with the bacterium of going out take out Cordyceps sinensis in the test tube, cut off fruit body with the aseptic operation cutter from the polypide head, use again another aseptic operation cutter that sclerotium (polypide part) is cut into 1.5～2.0 mm thin slices (mung bean size), transfer on worm slurry medium test tube slant or flat board, if dull and stereotyped, density is every dull and stereotyped 5-6 sheet.
C, above-mentioned test tube or flat board are put into full-automatic incubator, (natural Hirsutella sinensis infects the bat moth larvae and begins soil temperature when the pin main body Intracavity between 5.6～9.2 ℃ for 6～9 ℃ of set temperatures, can reduce at low temperatures other miscellaneous bacteria disturbs), illumination 50～100lux(is equivalent to indoor natural daylight), air humidity 55～60%, about oxygen content 20%, leave standstill and cultivate some days (tissue isolations 50～55 days, isolation of ascospores method 40～45 days), the visible bacterium colony of naked eyes.
Separate the culture that obtains with the present invention, described basically identical with Shen Nan English on the form, namely saw the white hypha that projection is level and smooth at 10～25 days, white becomes sepia after 30 days, and the likeness in form flower has pheomelanins around it, see long white hypha after 45 days, and being irregular cotton shape, the bacterium colony projection is brown or black in the base.By with GenBank in the contrast of aweto sequence number from FJ654206 to FJ654259, its similitude reaches 97.9%, is defined as Hirsutella sinensis.
Further illustrate the present invention below by embodiment, but do not limit the scope of the invention
Embodiment 1
Material source: the host bat moth larvae live body as the Cordyceps sinensis of natural medium, on November 5th, 2011, pick up from the state-owned pasture of Yushu Regions Yushu County, Qinghai Province, in the alpine meadow that height above sea level is 4000 meters; As the fresh Cordyceps sinensis of provenance, on May 31st, 2012, pick up from the Tibetan pasture of Yushu Regions Zhiduo County, Qinghai Province, in the alpine meadow that height above sea level is 4500 meters.
Hirsutella sinensis isolation of ascospores method:
1, at the scene that gathers fresh Cordyceps sinensis, a selection wherein fruit body is lacked (long 3cm), polypide sturdy (long 3.5cm, diameter 6.5mm), can be seen that pregnant adularescent ascospore of ascus overflows by naked eyes, kraft paper bag with the bacterium of going out entangles its fruit body at once, and places incubator;
2, the material that gathers is transported to the laboratory, put at once 0-4 ℃ refrigerator, take out this fresh Cordyceps sinensis after 5 days in the laboratory, white, wire ascospore on the brown paper are taken off, namely, the ox-hide scraps of paper with sterile scissors clip surface wire ascospore comparatively dense are stored in the PE box of the bacterium of going out.
3, select the host bat moth larvae live body of the Cordyceps sinensis of 5 3.5cm in the laboratory, be soaked in 75 ℃ of alcohol 3min, change clothes 3 times with sterile water.The bat moth larvae that to sterilize again cuts the enteron aisle of head, crust and black, collect, pull an oar with bar Ma Tianshou spring natural mineral water again, little adjust pH 6～6.5, adding 2%(percentage by weight) agar, make the test tube slant, wait to solidify namely as the natural medium that separates Hirsutella sinensis.
4, use the aseptic inoculation ring, under aseptic condition, picking wire ascospore (not being the polypide tissue) is directly transferred on worm slurry medium slant, all places 8 ℃ of incubators to cultivate.Can see the bacterium colony of the about 2cm of diameter in 45 days, the surface covers with undeveloped canescence mycelia, is black in the base.
The Hirsutella sinensis tissue isolation:
1, with aseptic water (about 10 ℃) and sterile razor blade hymeniderm (tunicle, the adhesive of mycelia and the soil) stripping on one fresh Cordyceps sinensis surface is washed off, exposed the yellow polypide to brown color of breast (rice).
2, then Chinese caterpillar fungus is placed a 200ml beaker that had gone out bacterium, carry out the accurate timing of surface sterilization 12 min(stopwatches with 0.1% mercury chloride (percentage by weight)), tweezers with the bacterium of going out press from both sides out Chinese caterpillar fungus again, immersion fills in the 200ml beaker of the bacterium of going out of about 100ml sterile water, washs 20 seconds.Tweezers with the bacterium of going out press from both sides out Chinese caterpillar fungus again, immerse in another 200ml beaker of the bacterium of going out that fills about 100ml sterile water, and above-mentioned washing step repeats, and share the about 1000ml of sterile water.
3, with isolation of ascospores method 3, but different be that test tube has changed flat board into.
4, will be the Chinese caterpillar fungus of flush away surface chlorination mercury wipe solid carbon dioxide with sterile gauze and divide, place the culture dish of the bacterium of going out, tweezers with the bacterium of going out are clamped, the aseptic operation cutter cuts off fruit body from the polypide head, with another aseptic operation cutter polypide is cut into 1.8 mm thin slices again, transfers on worm slurry culture medium flat plate, 4 of every flat boards, totally three, all place 9 ℃ of incubators to cultivate.Can see that pitchy secretion and a small amount of canescence mycelia were arranged in 15 days, can see the bacterium colony of the about 1cm of diameter in 50～55 days, the surface covers with undeveloped canescence mycelia, is brown or black in the base.
Embodiment 2
Material source: with embodiment 1
Hirsutella sinensis isolation of ascospores method:
1, at the scene that gathers fresh Cordyceps sinensis, a selection wherein fruit body is lacked (long 2.5cm), polypide sturdy (long 3.5cm, diameter 7mm), can be seen that pregnant of ascus is fuller by naked eyes, kraft paper bag with the bacterium of going out entangles its fruit body, and places incubator;
2, the material that gathers is transported to the laboratory, put at once 0-4 ℃ refrigerator, take out this fresh Cordyceps sinensis after 15 days in the laboratory, can see adularescent filiform on the brown paper, ascospore can be taken off, the ox-hide scraps of paper with sterile scissors clip surface wire ascospore comparatively dense are stored in the PE box of the bacterium of going out.
3, select the host bat moth larvae live body of the Cordyceps sinensis of 7 3.0cm in the laboratory, be soaked in 75 ℃ of alcohol 3min, change clothes 3 times with sterile water.The bat moth larvae that to sterilize again cuts the enteron aisle of head, crust and black, again with bar Ma Tianshou spring natural mineral water collect, making beating, little adjust pH 6～6.5 adds the 2%(percentage by weight) agar, make the test tube slant, wait to solidify namely as the medium that separates Hirsutella sinensis.
4, use the aseptic inoculation ring, under aseptic condition, picking wire ascospore is transferred on worm slurry medium slant, totally two, all places 7 ℃ of incubators to cultivate.Can see the bacterium colony of the about 2cm of diameter in 40 days, the surface covers with undeveloped canescence mycelia, is black in the base.
The Hirsutella sinensis tissue isolation:
1, with embodiment 1.
2, then Chinese caterpillar fungus is placed a 200ml beaker that had gone out bacterium, carry out the accurate timing of surface sterilization 10 min(stopwatches with 0.1% mercury chloride (percentage by weight)), tweezers with the bacterium of going out press from both sides out Chinese caterpillar fungus again, immersion fills in the 200ml beaker of the bacterium of going out of about 100ml sterile water, washs 20 seconds.Tweezers with the bacterium of going out press from both sides out Chinese caterpillar fungus again, immerse in another 200ml beaker of the bacterium of going out that fills about 100ml sterile water, and above-mentioned washing step repeats, and share the about 1000ml of sterile water.
3, with isolation of ascospores method 3, but different be that test tube has changed flat board into.
4, will be the Chinese caterpillar fungus of flush away surface chlorination mercury wipe solid carbon dioxide with sterile gauze and divide, place the culture dish of the bacterium of going out, tweezers with the bacterium of going out are clamped, the aseptic operation cutter cuts off fruit body from the polypide head, with another aseptic operation cutter polypide is cut into 1.8 mm thin slices again, transfers on worm slurry culture medium flat plate, 4 of every flat boards, totally two, all place 8 ℃ of incubators to cultivate.Can see that pitchy secretion and a small amount of canescence mycelia were arranged in 15 days, can see the bacterium colony of the about 1cm of diameter in 50～55 days, the surface covers with undeveloped canescence mycelia, is brown or black in the base.
Above embodiment all adopts Jintan, the Jiangsu Province 150B_250C of experimental instrument factory digital display illumination box.The parameters such as this incubator can A.T.C, air, illumination.
Claims (3)
1. the ecosystem partition method of an aweto Hirsutella sinensis may further comprise the steps and condition:
A, provenance are the fresh Cordyceps sinensis of maturation that comes from the Qinghai Yushu Regions, will keep the soil with diameter 10～12cm, dark 8～12cm around the fruit body during collection; Medium derives from the live body of the host bat moth larvae of Cordyceps sinensis, select white and moist, healthy and strong, bat moth larvae 4～5 length of times, that live, during collection in time with rinsed with sterile water and store; The storage temperature of live body after collection of the host bat moth larvae of described Cordyceps sinensis and Cordyceps sinensis is 0～4 ℃, and the storage time is 0～15 day;
B, provenance transferred cultivate on medium, parameter is controlled at 6～9 ℃ of temperature, pH value 6～6.5, illumination 50～100lux, air humidity 55～60%, oxygen content 20% during cultivation.
2. the ecosystem partition method of aweto Hirsutella sinensis according to claim 1 is characterized by further comprising the steps of and condition:
A, described provenance should in time use the kraft paper bag of the bacterium of going out to entangle the cordyceps sporophore part when gathering, the ascospore that the fruit body of described provenance is being launched separates, when namely on seeing brown paper, being attached with white, wire ascospore, with the ox-hide scraps of paper at wire ascospore place, sterile scissors clip surface, be stored in the PE box of the bacterium of going out for subsequent use;
B, the whole piece worm of bat moth larvae live body is immersed in the 75 degree alcohol 3 minutes, changes clothes 3 times with sterile water again; The bat moth larvae of sterilizing is cut the enteron aisle of head, crust and black, and again making beating with the little adjust pH to 6 of bar Ma Tianshou spring natural mineral water～6.5, adds 1.8～2.2%(wt%) agar, makes test tube slant or flat board, solidifies as medium;
C, cultivation: use the aseptic inoculation ring, the white wire ascospore on the picking ox-hide scraps of paper is transferred on described medium test tube slant or flat board, and puts into incubator and cultivated 40～45 days.
3. the ecosystem partition method of aweto Hirsutella sinensis according to claim 1 is characterized by further comprising the steps of and condition:
A, with the polypide part of described provenance, at first fall its surperficial hymeniderm with 9～11 ℃ of sterile water wash, then, with concentration 0.1% mercury chloride they were carried out surface sterilization 10～15 minutes, then, with sterile water wash 5～7 times, be stored in the test tube of the bacterium of going out;
B, the whole piece worm of bat moth larvae live body is immersed in the 75 degree alcohol 3 minutes, changes clothes 3 times with sterile water again; The bat moth larvae of sterilizing is cut the enteron aisle of head, crust and black, and again making beating with the little adjust pH to 6 of bar Ma Tianshou spring natural mineral water～6.5, adds 1.8～2.2%(wt%) agar, makes test tube slant or flat board, solidifies as medium;
C, cultivation: the tweezers with the bacterium of going out take out Cordyceps sinensis from test tube, cut off fruit body with the aseptic operation cutter from the polypide head, with another aseptic operation cutter polypide partly is cut into 1.5～2.0 mm thin slices again, transfer on medium test tube slant or flat board, and put into incubator and cultivate, 6～9 ℃ of set temperatures, illumination 50～100lux, air humidity 55～60%, oxygen content 20% was cultivated 50～55 days.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103392513A (en) * | 2013-08-14 | 2013-11-20 | 西藏现代农业有限公司 | Ferment of aweto mycelia and application of ferment |
| CN103444435A (en) * | 2013-09-05 | 2013-12-18 | 东方中科生命科学有限责任公司 | Method for breeding high-quality cordyceps sinensis strain |
| CN104904496A (en) * | 2015-06-18 | 2015-09-16 | 上海善力健生物科技有限公司 | Preparation method for cordyceps sinensis mycelium |
| CN105670937A (en) * | 2016-01-07 | 2016-06-15 | 广东东阳光药业有限公司 | Method for acquiring high quality Hirsutella sinensis strain |
| CN106010978A (en) * | 2016-05-25 | 2016-10-12 | 浙江农林大学 | Culture method of cordyceps sinensis anamorphic hirsutella sinensis pure strain |
| CN110447463A (en) * | 2019-08-12 | 2019-11-15 | 江门市山海堂虫草有限公司 | A kind of preparation method of cordyceps sporophore |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103392513A (en) * | 2013-08-14 | 2013-11-20 | 西藏现代农业有限公司 | Ferment of aweto mycelia and application of ferment |
| CN103444435A (en) * | 2013-09-05 | 2013-12-18 | 东方中科生命科学有限责任公司 | Method for breeding high-quality cordyceps sinensis strain |
| CN103444435B (en) * | 2013-09-05 | 2015-01-14 | 东方中科生命科学有限责任公司 | Method for breeding high-quality cordyceps sinensis strain |
| CN104904496A (en) * | 2015-06-18 | 2015-09-16 | 上海善力健生物科技有限公司 | Preparation method for cordyceps sinensis mycelium |
| CN105670937A (en) * | 2016-01-07 | 2016-06-15 | 广东东阳光药业有限公司 | Method for acquiring high quality Hirsutella sinensis strain |
| CN106010978A (en) * | 2016-05-25 | 2016-10-12 | 浙江农林大学 | Culture method of cordyceps sinensis anamorphic hirsutella sinensis pure strain |
| CN110447463A (en) * | 2019-08-12 | 2019-11-15 | 江门市山海堂虫草有限公司 | A kind of preparation method of cordyceps sporophore |
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